7. Project Summary/Abstract. Schizophrenia (SZ) is a devastating psychiatric disorder with limited treatment options. Disruptions in cortical circuitry are a key feature of SZ pathology and pathophysiology. Reduced dendritic spine density (DSD) in cortical layer 3 is among the most consistently observed cortical circuit abnormalities in postmortem studies of SZ, affecting multiple brain regions including the superior temporal gyrus (STG). Reduced DSD is thought to underlie multiple symptom domains in SZ including auditory processing deficits that impair social cognition and auditory hallucinations, however, the mechanisms that contribute to reduced DSD are poorly understood. DNA methylation (DNAm), the addition of a methyl group to a cytosine nucleotide, is a regulator of gene transcription. Given that (1) DNAm is altered in the brains of SZ subjects and (2) DNAm is altered in other contexts characterized by DSD abnormalities, DNAm is a strong candidate mechanism for reduced DSD in SZ. Despite evidence suggesting a role for DNAm in regulation of DSD, the relationship between DNAm and reduced DSD in SZ has not previously been explored. We propose studies to test the hypothesis that reduced DSD in SZ results, in part, from the altered transcription of multiple genes caused by alterations in DNAm. First, we will assess genome-wide, site-specific DNAm selectively in layer 3 of STG in a large SZ-NPC cohort for which DSD has already been characterized. Then, we will hone in on the DNAm-DSD correlations, and the DNAm-gene transcription relationships that may mediate those correlations, in STG layer 3 PYR neurons. Finally, we will test the causal relationship between DNAm and DSD by using the CRISPR/Cas9 system to alter candidate gene DNAm in a genome-region-specific manner and measure DSD in neuron cultures. To compliment this research project, I have developed an innovative, comprehensive, and multidisciplinary training plan to facilitate my transition to independent investigator. Upon completion of the proposed research and training plans, I will be an expert in psychiatric epigenetics, generally, and in applying cutting-edge approaches to the study of dendritic spine pathology in SZ, specifically. Few researchers have the necessary background and training to connect the multiple levels of investigation? clinical observation/behavior, circuits, neurons, transcript expression, epigenetic modifications, and genetic code?necessary to make innovative and clinically-relevant contributions to understanding the epigenetic mechanisms of psychiatric disorders. It is with this unique combination of background and training, that I will establish my independent, NIH-funded lab and submit for R01 funding in year 3 of my K23 award period.